Oncofetal antigen/immature laminin receptor antibodies for diagnostic and clinical applications

ABSTRACT

The present invention relates to antibodies against Oncofetal Antigen/immature Laminin receptor protein (OFA/iLRP) that can be used singly or in conjunction to detect or treat OFA/iLRP-related diseases. More specifically, the antibodies can be used for several purposes including: (i) detecting and measuring OFA/iLRP in different biofluids; and (ii) using OFA/iLRP with an antibody directed against the monomeric form and its associated diseases.

The present application claims the benefit of the filing date of U.S.Provisional Application No. 61/163,810 filed Mar. 26, 2009, thedisclosure of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates in general to the oncofetalantigen/immature laminin receptor protein (OFA/iLRP). More specifically,the invention provides antibodies that can be used to detect and treatOFA/iLRP-related diseases.

BACKGROUND OF THE INVENTION

The initial characterization of oncofetal antigen/immature lamininreceptor protein (OFA/iLRP) was done by three independent groups, whichstudied oncofetal antigen or laminin receptor [1-3]. OFA/iLRP is ahighly conserved protein that is over-expressed in a range of differentcancers and has a dual function as ribosomal protein p40 [4-27]. TheOFA/iLRP protein is comprised of a single polypeptide chain of 295 aminoacids and has a molecular weight of about 37-44 kDa. The structure ofOFA/iLRP has recently been elucidated to 2.15 Å [28]. The mature form ofthe laminin receptor (LRP) appears to be a dimer of acetylated OFA/iLRP,with a molecular weight of 67 kDa. The structure showed that the regionbetween amino acids 112 to 140 of OFA/iLRP is involved in dimerization[28] of OFA/iLRP for forming the LRP. Although the 67 kDa LRP is on manynormal cells as well as on tumor cells, there appears to be apreferential expression of the OFA/iLRP by fetal and tumor cells. Thus,the expression pattern makes OFA/iLRP a possible candidate protein tosensitize the immune system for the treatment of cancer and otherdiseases [6]. Antibodies specific for OFA/iLRP may also be used for thedetection, diagnosis, and treatment of diseases known to be related toOFA/iLRP mis-expression.

The initial work on OFA/iLRP antibodies falls under two separate fields,the oncofetal antigen or the laminin receptor sides. The initial reportof monoclonal antibodies against OFA/iLRP was found the same year forboth the embryonic/fetal antigen and the laminin receptor [29, 30]. Theantibodies developed against embryonic or fetal antigen reacted with a44 kDa protein under denaturing conditions [30]. Antibodies previouslydeveloped against the laminin receptor had different biologicalactivities based on the location of antibody binding [29]. One regionthat had biological activity of blocking laminin binding was recognizedby monoclonal IgM antibody. The epitope that the monoclonal IgM antibodyrecognized was TEDWSAQPATEDWSA [26]. Studies on the 44 kDa OFA showedthat the IgM monoclonal antibody (MAb 115) can be used for westernblots, flow cytometry, and possibly oncogenicity testing [31]. However,since this antibody was not designed specifically against the OFA|OFAdimerization region, it reacts with both OFA/iLRP and LRP [31]. Thismonoclonal antibody was used for immunohistochemistry and proteinpurification of the OFA/iLRP or LRP [12, 32]. A different antibody wasdeveloped from peptides that detected the 67 kDa laminin receptor andshowed increased laminin receptor expression in breast cancer [5, 33].Several published manuscripts describe the use of OFA/iLRP antibodies,while looking for autoimmune antibodies [6, 9, 10, 16, 19, 20, 25, 27,34-36].

Since OFA/iLRP is associated with a range of different diseases, thereis a need to develop diagnostic and clinical antibody applications. Todate, there have been only a few attempts to develop a targeted antibodyusing peptides and this has been limited to one published report [5].The region used in the published report produced antibodies that wasreported to react with both the OFA/iLRP and laminin receptor [5, 25].Thus, a need exists to develop antibodies that are specific to OFA/iLRP,which can be used alone or in conjunction with other antibodies, todevelop a range of different clinical, diagnostic, and/or veterinaryapplications. The development of antibodies in pairs, one that canrecognize OFA/iLRP and one that can recognize both OFA/iLRP and LRP,allows for the development of several tests that can be use to treat,diagnosis or act as a reagent in OFA/iLRP diseases in all species due tothe conserved nature of the protein.

SUMMARY OF THE INVENTION

One aspect of the present invention provides an isolated antibody thatspecifically binds to a region of the oncofetal antigen/immature lamininreceptor protein (OFA/iLRP), wherein the region is immunogenic. In oneembodiment, the immunogenic regions of OFA/iLRP include, but are notlimited to, polypeptide sequences of (a) FFREPRLLVVTDPR, (b)VTDPRADHQPLTE, (c) YRDPEEIEKEEQ, or (d) FPTEDWSAQPATED. In anotherembodiment, the isolated antibody of the present invention is apolyclonal or monoclonal antibody, including but not limited to,monoclonal antibody 2C6 or 3G7. In further another embodiment, theimmunogenic region is located in the dimerization region of OFA/iLRP.

The antibodies of the present invention are produced by immunizingagainst immunogenic regions of OFA/iLRP. The antibodies can recognizeboth the full-length OFA/iLRP protein and the immunogenic regions ofOFA/iLRP that are used to produce the antibodies of the presentinvention. In one embodiment, the antibodies are specific for OFA/iLRP.In an alternative embodiment, the antibodies recognize both OFA/iLRP andmature LRP.

The antibodies of the invention can be used in different methods fordetecting proteins or cancers related to OFA/iLRP.

In one embodiment, the antibodies are used in a method for detectingOFA/iLRP in a sample. The method comprises:

-   -   (a) contacting the sample with first and second antibodies that        specifically bind to a region of the oncofetal antigen/immature        laminin receptor protein (OFA/iLRP) respectively, wherein at        least one of the antibodies is specific for OFA/iLRP;    -   (b) allowing the antibodies to bind to OFA/iLRP and form a        sandwich with OFA/iLRP; and    -   (c) detecting the expression of OFA/iLR in the sample using the        antibody specific for OFA/iLRP.

In one embodiment, one of the antibodies may bind to both OFA/iLRP andmature LRP, and act as a capture antibody. Another antibody may bespecific for OFA/iLRP and acts as a detection antibody.

In another embodiment, the antibodies of the invention are used in amethod of detecting cancer in a sample. The method comprises:

-   -   (a) contacting the sample with antibodies specific for OFA/iLRP;    -   (b) contacting the sample with a biotinylated secondary        antibody; and    -   (c) detecting OFA/iLRP in the sample using streptavidin,

wherein the detection of OFA/iLRP in the sample is indicative of cancer.

In, a further embodiment, the invention provides a method of determiningthe amount of OFA/iLRP in a sample comprising:

-   -   (a) conjugating an antibody specific for OFA/iLRP to a        fluorophore;    -   (b) contacting the conjugated antibody in a sample; and    -   (c) determining the amount of OFA/iLRP in the sample using        fluorescent polarization.

Antibodies of the present invention may also be used in a method ofdetermining the amount of OFA/iLRP positive cancer cells in a bloodsample. The method comprises:

-   -   (a) contacting a blood sample with an antibody specific for        OFA/iLRPl and    -   (b) determining the amount of OFA/iLRP in the sample using flow        cytometry.

Antibodies of the invention may also be used in a method of treating aOFA/iLRP positive cancer. The method comprises administering an amountof antibodies specific for OFA/iLRP to a subject with the OFA/iLRPpositive cancer that is sufficient to ameliorate the cancer-relatedsymptoms. In one embodiment, the antibodies are linked to colloidshaving anti-cancer properties or are conjugated with chemotherapeuticagents or protein.

The present invention also provides methods of detecting a OFA/iLRPpositive cancer in a subject. The method comprises:

-   -   (a) conjugating antibodies specific for OFA/iLRP to a radiopaque        dye;    -   (b) administering the conjugated antibody to a subject; and    -   (c) detecting the conjugated antibody using an x-ray,

wherein the detection of OFA/iLRP on the x-ray is indicative of cancer.

Another aspect of the present invention provides a pharmaceuticalcomposition comprising an antibody of the present invention. Thecomposition may include a pharmaceutical carrier.

The above-mentioned and other features of this invention, and the mannerof obtaining and using them, will become more apparent, and will be bestunderstood by reference to the following description, taken inconjunction with the accompanying drawings. The drawings depict onlytypical embodiments of the invention and do not therefore limit itsscope.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Titration of OFA/iLRP polyclonal serum against BSA conjugateOFA/iLRP peptides. Detection of antibody antigen reaction used abiotinylated universal secondary antibody mixture. Absorbance wasmeasured on a SpectraMax and data was analyzed using Prism Graph.

FIG. 2. Titration of OFA/iLRP polyclonal serum against OFA/iLRP.Detection of OFA/iLRP antibody antigen interaction using a biotinylatedsecondary antibody. Prism Graph of the Absorbance read at A620 on aBeckman DTX 880.

FIG. 3. Dilution curve of monoclonal antibodies 2C6 and 3G7 run on anindirect ELISA against OFA/iLRP.

FIG. 4. Sandwich ELISA of rOFA/iLRP to determine the reactivity ofmonoclonal antibodies (2C6 and 3G7). Raw data was plotted on a semi-loggraph and cure fitting was done using the 4-parameter logistic followingstandard protocols (SoftMax Pro 4.3.1 LS).

FIG. 5. Fluorescent polarization of OFA/iLRP. (A) Raw data was plottedon a log x-axis. (B) Curve fit data using four-parameter logistic(r²=0.975) n=2.

FIG. 6. IHC staining of human invasive ductal carcinoma, moderatelydifferentiated, T2N1M0 using 3G7 monoclonal antibody for the primaryantibody reaction. The very dark regions are where cells are expressinglarge amounts of OFA/iLRP and a thick precipitate was deposited.

FIG. 7. The effect of 2C6 and 3G7 antibodies on cell viability wasmeasured using CellTiter Blue. The decreased viability caused by theaddition of antibodies is indicated by a lower fluorescent signal. Theeffect of the antibodies and requirement for laminin was determined bygrowing on either laminin/entactin coated or untreated plates.

DETAILED DESCRIPTION OF THE INVENTION

It was a surprise discovery of the present invention that immunogenicpeptides may be derived from putative isotope regions of OFA/iLRP, andused to generate antibodies against specific regions of OFA/iLRP thatwere not previously discovered.

Accordingly, one aspect of the present invention is directed to anisolated antibody which specifically binds to a region of the oncofetalantigen/immature laminin receptor protein (OFA/iLRP), wherein the regionis immunogenic. For the purpose of the present invention, an “isolated”antibody is one which has been identified and separated, and/orrecovered from a component of its natural environment.

In one embodiment of the present invention, the antibody is specific forOFA/iLRP. An antibody is specific for OFA/iLRP if it preferentiallybinds to OFA/iLRP, not the 67 kDa form of the laminin receptor under awell-known standard antibody-binding condition. Examples of such anantibody include, but are not limited to, 3G7. In a differentembodiment, the antibody of the present invention may recognize bothOFA/iLRP and the 67 kDa form of the laminin receptor. Examples of suchan antibody include, but are not limited to, 2C6. In a furtherembodiment, the antibody of the invention bind to the dimerizationregion of OFA/iLRP. For the purpose of the present invention, thedimerization region is a region between amino acids 112 to 140 ofOFA/iPRP full-length protein that are involved in dimerization [28] ofOFA/iLRP.

The antibodies of the present invention can recognize full lengthOFA/iLRP proteins. They can also recognize specific regions of OFA/iLRPthat are immunogenic, particularly the dimerization region. For thepurpose of the present invention, a region of OFA/iLRP is immunogenic ifpolypeptides derived from that region can provoke an immune response andcan be used to produce the antibodies of the present invention. Forexample, antibodies generated by the four immunogenic peptides listed inTable 2 can recognize the full-length OFA/iLRP protein, their respectiveimmunogenic peptides, and the regions of OFA/iLRP comprising thosepeptides.

For the purpose of the present invention, an “antibody” is animmunoglobulin molecule capable of specific binding to a target, such asa receptor, carbohydrate, polynucleotide, lipid, polypeptide, etc.,through at least one antigen recognition site, located in the variableregion of the immunoglobulin molecule. As used herein, the termencompasses not only intact polyclonal or monoclonal antibodies, butalso fragments thereof (such as Fab, Fab′, F(ab′).sub.2, Fv), singlechain (ScFv), mutants thereof, fusion proteins comprising an antibodyportion, and any other modified configuration of the immunoglobulinmolecule that comprises an antigen recognition site. An antibodyincludes an antibody of any class, such as IgG, IgA, or IgM (orsub-class thereof), and the antibody need not be of any particularclass. Depending on the antibody amino acid sequence of the constantdomain of its heavy chains, immunoglobulins can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. Theheavy-chain constant domains that correspond to the different classes ofimmunoglobulins are called alpha, delta, epsilon, gamma, and mu,respectively. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well known.

The antibody of the present invention is further intended to includebispecific, multispecific, single-chain, and chimeric and humanizedmolecules having affinity for a polypeptide conferred by at least oneOFA/iLRP region of the antibody. Antibodies of the present inventionalso include single domain antibodies which are either the variabledomain of an antibody heavy chain or the variable domain of an antibodylight chain. Methods of making domain antibodies comprising either thevariable domain of an antibody heavy chain or the variable domain of anantibody light chain, containing three of the six naturally-occurringcomplementarity-determining regions from an antibody, are also known inthe art. See, e.g., Muyldermans, Rev. Mol. Biotechnol., 74:277-302,2001.

In one embodiment, the antibodies developed against OFA/iLRP aremonoclonal antibodies. Monoclonal antibodies, such as 2C6 and 3G7, canalso recognize the full length OFA/iLRP protein, their respectiveimmunogenic peptides, and the regions of OFA/iLRP comprising thosepeptides. However, monoclonal antibody 3G7 does not recognize the matureform of LRP (the dimer form), whereas monoclonal antibody 2C6 does.Although, high doses of 3G7 antibody may disrupt the OFA/iLRP dimer.

As used herein, the term “monoclonal antibody” refers to an antibody ofsubstantially homogeneous antibodies, i.e., the individual antibodiescomprising the population are identical except for possiblenaturally-occurring mutations that may be present in minor amounts.Monoclonal antibodies are generally highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations, which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies to be used inaccordance with the present invention may be made by recombinant DNAmethods such as described in U.S. Pat. No. 4,816,567. The monoclonalantibodies may also be isolated from phage libraries generated using thetechniques described in McCafferty et al., 1990, Nature, 348:552-554,for example.

The antibodies of the present invention are produced by immunizingagainst peptides based on selected criteria. The selection of thepeptides and the selection criteria are fully disclosed in the patentapplication entitled “Oncofetal Antigen/Immature Laminin ReceptorPeptides For The Sensitization Of Dendritic Cells For Cancer Therapy”which is concurrently filed with the present application, the relevantcontent of which is fully incorporated herein. In general, a polypeptideor peptide comprising at least a part, i.e., the whole or a part, of theamino acid sequence of OFA/iLRP, may be used as an antigen as long as itcan elicit specific antibody response wherein the antibodies sogenerated can recognize both the full length OFA/iLRP and theimmunogenic peptides used therein. According to embodiments of thepresent invention, immunogenic peptides are generated based on differentputative epitope regions of OFA/iLRP. In order to increasereproducibility and the chances of a multi-functional antibody againstOFA/iLRP, either to be used alone or in conjunction with the antibodyagainst the dimerization region of OFA/iLRP, preferably, antibodies aredeveloped against the regions or epitopes listed in Table 1. In oneembodiment, the following four peptides are used for generatingantibodies of the present invention: 1) FREPRLLVVTDPR, 2) VTDPRADHQPLTE,3) YRDPEEIEKEEQ, 4) FPTEDWSAQPATED.

The peptides of the present invention may be prepared by chemicalsynthesis or biochemical synthesis using Escherichia coli or the like.Methods well known to those skilled in the art may be used for thesynthesis.

When the peptide of the invention is chemically synthesized, methodswell known in the field of peptide synthesis may be used. For example,such methods as the azide method, the acid chloride method, the acidanhydride method, the mixed acid anhydride method, the DCC method, theactive ester method, the carbodiimidazole method and theoxidation-reduction method may be enumerated. Either solid phasesynthesis or liquid phase synthesis may be used. A commercial peptidesynthesizer (e.g., Shimadzu PSSM-8) may also be used.

After the reaction, the peptide of the invention may be purified by acombination of conventional purification methods such as solventextraction, distillation, column chromatography, liquid chromatography,or re-crystallization.

The peptides of the invention may be modified to increase itsimmunogenic response. In one embodiment, alterations of the peptides mayinclude a cysteine residue at either end that allow for conjugation toKeyhole Limpet hemocyanin, ovalbumin, serum albumin, or other conjugatesused to increase a peptide immunogenic response. Table 2 lists theexamples of modified peptides for conjugation.

The peptides can be used in a range of different organisms to createantibodies. The antibodies can also be cloned to generate a range ofdifferent recombinant antibodies based on well-known proteintechnologies. One of the objectives of the present invention is todevelop antibodies directed against specific regions of OFA/iLRP byimmunizing against the peptides of the present invention instead ofimmunizing against the full-length protein, and selecting antibodiesbased on their immuno-specificities against specific regions ofOFA/iLRP.

The monoclonal antibodies of the present invention can be generatedusing methods known in the art. For example, they may be generated byculturing the hybridoma cells, and the antibodies secreted by thehybridoma cells may further be isolated or purified. Antibodies may beisolated or purified from the culture medium or ascites fluid byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose.RTM., hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

The antibodies of the invention can also be made by recombinant DNAmethods, such as those described in U.S. Pat. Nos. 4,816,567 and6,331,415, which are hereby incorporated by reference, for example, DNAencoding the monoclonal antibodies of the invention can be readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of murine antibodies). The hybridomacells of the invention serve as a preferred source of such DNA. Onceisolated, the DNA can be placed into expression vectors, which are thentransfected into host cells, such as simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of monoclonal antibodiesin the recombinant host cells. The DNA also can be modified, forexample, by substituting the coding sequence for human heavy and lightchain constant domains in place of the homologous murine sequences (U.S.Pat. No. 4,816,567) or by covalently joining to the immunoglobulincoding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptidecan be substituted for the constant domains of an antibody of theinvention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

The antibodies of the present invention can be used for a range ofdifferent functions. In one embodiment, antibodies of the presentinvention may be used to detect malignant tissue through conjugationwith chemical, radiological, and nuclear adjuvants. Additionally, theseantibodies may be used as therapeutically to provide passive immunity,increased immune response in its native or altered or if conjugated toa: chemical, radiological “seed” source, microelectrical devices, silvercolloid, gold, titanium dioxide colloid, biopolymer colloid (i.e.,starch, collagen, agarose, etc.), peptide sequence or protein sequence,to aid in the targeted destruction of malignant cells. Due to the highlyconserved nature of OFA/iLRP, the antibodies can also be used as areagent and should cross-react with all organisms that express theprotein.

Accordingly, another aspect of the present invention providespharmaceutical compositions comprising antibodies or polypeptidesdescribed herein. The antibodies or polypeptides of the compositions maybe used alone or conjugated to chemical, radiological “seed” source,microelectrical devices, silver colloid, gold, titanium dioxide colloid,biopolymer colloid (i.e., starch, collagen, agarose, etc.), peptidesequence, or protein sequence, that can aid in the targeted destructionof malignant cells.

The compositions may also comprise a pharmaceutically acceptable carrieror excipients. Pharmaceutically acceptable excipients are known in theart, and are relatively inert substances that facilitate theadministration of a pharmacologically-effective substance. For example,an excipient can give form or consistency, or act as a diluent. Suitableexcipients include, but are not limited to, stabilizing agents, wettingand emulsifying agents, salts for varying osmolarity, encapsulatingagents, buffers, and skin penetration enhancers. Excipients as well asformulations for parenteral and nonparenteral drug delivery are setforth in Remington, The Science and Practice of Pharmacy 20th Ed. MackPublishing (2000).

The composition of the invention is formulated to be compatible with itsintended route of administration. Examples of routes of administrationinclude parenteral, e.g., intravenous, intradermal, subcutaneous, oral(e.g., inhalation), transdermal (topical), transmucosal, and rectaladministration. Solutions or suspensions used for parenteral,intradermal, or subcutaneous application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates; and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes, or multiple dose vials made of glass or plastic.

Compositions suitable for injectable use include sterile aqueoussolutions (where water soluble) or dispersions, and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersion. For intravenous administration, suitable carriers includephysiological saline, sterile water, Cremophor EL™ (BASF, Parsippany,N.J.), or phosphate buffered saline (PBS). In all cases, the compositionmust be sterile and should be fluid to the extent that easysyringability exists. It should be stable under the conditions ofmanufacture and storage, and must be preserved against the contaminatingaction of microorganisms such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquidpolyetheylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion, and by the use of surfactants. Prevention of theaction of microorganisms can be achieved by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol,ascorbic acid, thimerosal, and the like. In many cases, it will bepreferable to include isotonic agents, for example, sugars, polyalcoholssuch as manitol, sorbitol, or sodium chloride in the composition.Prolonged absorption of the injectable compositions can be brought aboutby including in the composition an agent which delays absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating thecompounds in the required amounts in an appropriate solvent with one ora combination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the compounds into a sterile vehicle which contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying, which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, thecompounds can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches, and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compositions are delivered in theform of an aerosol spray from a pressurized container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the compounds are formulated into ointments,salves, gels, or creams as generally known in the art.

The compositions of the invention can also be prepared in the form ofsuppositories (e.g., with conventional suppository bases such as cocoabutter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the compositions are prepared with carriers that willprotect the compounds against rapid elimination from the body, such as acontrolled release formulation, including implants and microencapsulateddelivery systems. Biodegradable, biocompatible polymers can be used,such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid,collagen, polyorthoesters, and polylactic acid. Methods for preparationof such formulations will be apparent to those skilled in the art. Thematerials can also be obtained commercially from Alza Corporation andNova Pharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to infected cells with monoclonal antibodies to viral antigens)can also be used as pharmaceutically acceptable carriers.

It is advantageous to formulate oral or parenteral compositions indosage unit form for ease of administration and uniformity of dosage.“Dosage unit form,” as used herein, refers to physically discrete unitssuited as unitary dosages for the subject to be treated. Each unitcontains a predetermined quantity of active compound calculated toproduce the desired therapeutical effect in association with therequired pharmaceutical carrier.

The compositions of the invention can be included in a container, pack,or dispenser, together with instructions for administration to formpackaged products. Other active compounds can also be incorporated intothe compositions.

A further aspect of the present invention provides a method of using theantibodies and polypeptides of the present invention for detection,diagnosis, and monitoring of a disease, disorder, or conditionassociated with the epitope expression of OFA/iLRP, either increased ordecreased relative to a normal sample, and/or inappropriate expression,such as the presence of expression in tissues(s) and/or cell(s) thatnormally lack the epitope expression.

In some embodiments, the method comprises detecting the epitopeexpression in a sample obtained from a subject suspected of havingcancer, or any disease that is associated with OFA/iLRP. Preferably, themethod of detection comprises contacting the sample with an antibody orpolypeptide of the present invention under a condition that allows thebinding of the antibody to the epitope region of OFA/iLRP anddetermining whether the level of binding differs from that of a controlor comparison sample. The method is also useful to determine whether theantibodies or polypeptides described herein are an appropriate treatmentfor the patient.

For purposes of diagnosis, the polypeptide including antibodies can belabeled with a detectable moiety including, but not limited to,radioisotopes, fluorescent labels, and various enzyme-substrate labelsknown in the art. Methods of conjugating labels to an antibody are knownin the art.

In some embodiments, the polypeptides including antibodies of theinvention need not be labeled, and the presence thereof can be detectedusing a labeled antibody which binds to the antibodies of the invention.

The antibodies of the present invention can be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays. Zola, MonoclonalAntibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).

The antibodies and polypeptides can also be used for in vivo diagnosticassays, such as in vivo imaging. Generally, the antibody or thepolypeptide is labeled with a radionuclide (such as ¹¹¹In, ⁹⁹Tc, ¹⁴C,¹³¹I, ¹²⁵I, or ³H), so that the cells or tissue of interest can belocalized using immunoscintigraphy.

The antibody may also be used as a staining reagent in pathology usingtechniques well known in the art.

The invention will be further described by reference to the followingdetailed embodiments and examples. These embodiments and examples areprovided for the purposes of illustration only, and are not intended tolimit the scope of the invention described herein.

Development of Antibodies Against Specific Regions of OFA/iLRP

Antibodies are made against OFA/iLRP using a range of different peptidesthat are directed against a range of different putative epitope regionsof OFA/iLRP. Table 1 provides a list of such peptides. The peptides areconjugated to Keyhole Limpet Hemocyanin (KLH) following standardprotocols [37-39]. The KLH-OFA/iLRP peptides are used to immunize micefollowing standard protocols [37, 39]. After the third immunizationagainst the KLH-OFA/iLRP peptide, the serum isolated from mouse-tailbleed are screened against bovine serum albumin (BSA)conjugated-OFA/iLRP peptides using an indirect ELISA technique usingstandard protocols (FIG. 1 and Table 1). Briefly, OFA/iLRP was coated ona Nunc Star Immunosorp plate overnight at 4° C. The plate was washed andblocked 3 times with water. Mouse serum obtained via tail bleed wasdiluted in PBS-t with BSA and was incubated for 1 hour at room temp. Theplate was washed and the antibody antigen interaction was detected usingstandard methods with a biotinylated secondary antibody (FIGS. 1A-B).Absorbance was read at A620 on a Beckman DTX 880 and graphed using PrismGraph 5 (FIG. 1C). The bars show the mean of two independent experimentsfrom two animals per immunizing peptide. Positive control was coatedwith BSA and BSA antibodies. The negative control had no proteincoating.

In addition to peptide screening, the tail-bleeds are tested againstOFA/iLRP isolated using standard protocols [1-3, 5, 26, 29, 30].Briefly, full length OFA/iLRP is coated on a Nunc-Immuno Star MaxiSorpplate and diluted to 5 μg/ml in PBS with sodium azide for 2 hours at 37°C. The plates are washed three times in 18.2 megaohm water and blockedfor 1 hour at 25° C. using PBS-t with 5% dry milk powder. The plate iswashed 3 times with PBS-t. The serum from the tail bleed is diluted inblocking buffer (above) and incubated overnight at 4° C. The plate iswashed using PBS-t as above. The antibody-antigen complex is detectedusing a biotin goat anti-mouse antibody, diluted in blocking buffer, andincubated for 1 hour at 25° C. The plate is washed three times usingPBS-t. The secondary antibody is detected using Strepavidin-HorseradishPeroxidase (HRP), diluted in the blocking buffer, and incubated for 30min at 25° C. Plates are washed as above. The final detection stepfollows standard protocols using a Sigma FAST™ OPD system and read on aSpectraMax 384.

Antibodies Used in an Antibody-Sandwich ELISA to Detect OFA/iLRP

The expression of OFA/iLRP RNA and protein have been linked to moreaggressive forms of cancer [2, 3, 5, 6, 8, 10, 13, 15-17, 20, 21, 23,25, 26, 29, 40]. However, the protein work has primarily concentrated onthe 67 kDa form and immunohistochemistry. One of the objects of thepresent invention is to develop a standard sandwich ELISA where twoOFA/iLRP antibodies designed against distinct regions are used. Thecapture antibody that is specific for OFA/iLRP can be used to quantifythe amount of 37 kDa OFA/iLRP present, and capture antibodies outsidethis region can be used to quantify total OFA/iLRP present.

Briefly, the antibody-sandwich ELISA follows standard techniques asfollows. The capture antibody is coated on aNunc Star Immunosorb plate(or similar) which is then coated with a range of differentconcentrations of capture antibody against the FREPRLLVVTDRADHQPLTpeptide in carbonate buffer and incubated at 4° C. overnight. Theoptimum concentration of capture and detection antibody is determinedusing the “criss-cross” method of dilutions [39]. The plate is washedthree times in water and three times with PBS-t. Once washed, the plateis blocked for 1 hour using a standard blocking buffer. The blockedplate is washed three times with PBS-t. The soluble protein lysate istested with a standard blocking buffer and OFA/iLRP is incubated from 1hour at room temperature to overnight at 4° C. When the initialincubation is completed, the plate is washed 3 times with PBS-t. Todetect the OFA/iLRP capture antibody interaction, a secondary/detectionantibody is diluted in blocking buffer and incubated at room temperaturefor at least one hour. The plate is washed 3 times using PBS-t. Thedetection antibody is conjugated to biotin (or other methods). Thebiotin conjugate is detected using Strepavidin/Horseradish Peroxidase(HRP) or the equivalent. The strepavidin is diluted in an appropriateblock and incubated for 30 minutes at room temperature. The plate iswashed and the HRP enzymatic conjugate on the strepavidin is detectedusing the SIGMA FAST™ OPD system following standard protocols. Theamount of OFA/iLRP present is read on a SpectraMax 384 or theequivalent. The concentration of OFA/iLRP is back-calculated using thestandard curve generated from purified OFA/iLRP and multiplied by thedilution factor following standard curve fitting methods [37]. It mayalso be possible to detect the monomeric form of OFA/iLRP by using anon-specific capture antibody, followed by using a detection antibodythat is directed against the monomeric region. A combination of captureand detection antibodies against regions outside of the dimerizationregion can be used to determine the total amount of OFA/iLRP present inbiofluids or tissue lysates.

To detect the native dimeric form of OFA/iLRP, a standard ELISA is usedas above. However, instead of using an antibody developed against themonomeric region, the antibodies used are outside of this region, buthave a great enough distance from the detection antibody used above toallow for proper binding. The 67 kDa OFA/iLRP is purified followingstandard protocols [2, 3, 26, 29]. The size is verified as above, eitherusing native gel electrophoresis or size exclusion chromatography. Astandard ELISA is performed with a capture antibody against an epitopethat is different than FREPRLLVVTDRADHQPLT to capture the dimer, andthen the biotinylated detection antibody used in the monomer detectionis used. ELISA is processed following standard protocols [39]. This willdetermine the amount of dimer OFA/iLRP present in biofluids and tissuelysate. Additional information on the nature, metastatic load, and otherclinically relevant information may be obtained by the ratio ofmonomeric OFA/iLRP to total OFA/LRP.

Immunohistochemical Detection of OFA/iLRP

The antibodies listed above can be used for immunohistochemistry (IHC)as a method to detect cancer and possible cancer in situ. Similartechniques have been used to identify cancer, however, they detect the67 kDa protein [5, 17, 41]. IHC methods follow the standard protocolsthat have been customized for OFA/iLRP detection. The protocol issimilar to listed below:

Cut tissue is deparaffinized following standard protocols forhistological analysis of tissue, such as xylenes, followed by descendingconcentrations of ethanol, and into at least Type I Lab water. Antigenretrieval follows standard protocol, using Heat Induced EpitopeRetrieval (HIER) in 0.01 M citrate buffer adjusted to pH 6.0. Theepitope(s) can be retrieved using either direct heat method (hotplate)or an indirect heat method (microwave) [37]. Briefly, the deparaffinizedtissue sections are placed in a slide holder and placed in a stainingdish filled with citrate buffer at >90° C. The tissue is heated andmaintained >90° C. for around 10 minutes. The time of processing dependson the tissue type, tissue thickness, and a range of other factors. Onceproperly processed, the tissue is washed 3 times in phosphate bufferedsaline with 0.1% Tween-20 (PBS-t) following standard methods. The tissueis incubated with standard IHC blocking buffer (PBS-t with BSA or otherreagents used to decrease non-specific antibody/protein interactions) atroom temperature in a humid chamber. The slides are washed three timesin PBS-t. The OFA/iLRP antibodies are diluted appropriately in PBS-twith BSA or the equivalent and incubated with the processed tissue. Theslides are washed as above (3×PBS-t). The tissue is incubated withbiotinylated universal secondary antibody (or equivalent) to allow fordetection using a strepavidin-based system. The slides are washed asabove and the OFA/iLRP|Ab|2° antibody complex is detected using standardstrepavidin conjugated to either: Alkaline Phosphatase (AP), HorseradishPeroxidase (HRP), Quantum Dot, fluorophore, radio nucleotide, or otherdetection method [37, 39, 42]. The tissue is visualized followingstandard protocols.

Use of OFA/iLRP Antibodies for Fluorescent Polarization Quantification

The antibodies can be used for fluorescent polarization (FP) experimentsto determine the amount of OFA/iLRP present. Additionally, a ratio of FPof monomer versus dimer may be possible. FP can also be used todetermine the mobility of OFA/iLRP on the cell surface. Determining theuse of the antibodies in a fluorescent polarization experiment is basedupon previous work but is developed specifically for OFA/iLRPquantification [43-49]. In order to determine the use of OFA/iLRPantibodies, the monoclonal antibody is conjugated to a standardfluorophore and the labeled antibody can be removed from the free labelfollowing the SM-2 bio-beads protocol [50]. Once purified, the proteinconcentration and fluorescent incorporation can be determined as a ratioof protein to fluorophore. Once conjugated, the antibody can be storedappropriately and used later.

To test the ability of the antibody to be used in FP experiments, theantibody can be mixed with the appropriate amounts similar to thedetermination of the antibody concentration commonly used for the ELISAtechnique [39]. Briefly, a 96-well plate designed for fluorescence isused having a double dilution with the amount of OFA/iLRP protein alongone axis and the amount of labeled antibody along the other. Thedilutions take place in standard FP buffers and may contain a range ofdifferent additives to prevent non-specific binding. A prototypicalbuffer may contain the following: phosphate buffered saline, NP40,bovine serum albumin, bovine gamma globulin, glycerol, or other agentsto decrease background noise and increase specificity. Once mixed, theplate is placed in a Beckman DTX 880, and using the included FP(fluoroscein) filter, set to a read time of around 1 second per well orother, depending on the fluorophore used. Polarization values aremeasured in millipolarizations units (mP) calculated using the equation:mP=[(Is−IsB)−(Ip−IpB]/[(Is−IsB)+(Ip−IpB)]×1000 [47-49]. Initialexperiments generate dose response curves based upon the concentrationof the antibody and OFA/iLRP. After that, excess unlabeled antibody canbe used as a competitor to determine specificity and equilibrium.Additionally, the data generated from this can be used to back-calculatebinding analysis plots (i.e., Scatchard or similar), and in turn, usedas a method to determine the efficiency of antibodies. Once the initialconditions are determined, the FP method can be used to calculate theamount of OFA/iLRP present in different biofluids and tissue lysates.

Use in Flow Cytometric Analysis to Either Isolate/Separated OFA/iLRPPositive Cells or to Determine the OFA/iLRP Positive Cancer Cell Load inthe Blood

To determine the total cancerous load of circulating hematologic typecancers, or to determine metastatic load, flow cytometry may be used.Briefly, a cell suspension or peripheral blood is obtained. The cellsuspension is centrifuged 8 to 10 minutes at 300×g and the supernatantremoved. The cells are washed in flow cytometry staining buffer (HBBSwithout phenol red, 0.1% sodium azide, and 1% bovine serum albumin) onetime and resuspended at around 2×10⁷ cells/ml or 50 to 100 μl of thecell suspension on the bottom of a 96-well round bottom plate orequivalent. To the cell suspension, fluoroscein isothiocyanate (FITC) orequivalent labeled OFA/iLRP antibody diluted in staining buffer isadded. As a control for background, the cells are tested without anyantibody, with an unlabeled isotype control antibody or equivalent(excess unlabeled antibody or competitor peptide). Cell suspension andappropriately diluted antibody is incubated for 20 to 60 min on ice or4° C. with gentle mixing. The stained cell suspension is washed threetimes with 100 μl of staining buffer and centrifuged 3 to 10 min at 300to 500×g. After the final wash, the cells are suspended in around 400 μlof staining buffer and stored in the dark, covered at 4° C. untilanalyzed. Additional steps may include counterstaining with propidiumiodide to detect dead cells or the addition of a fixation step. Data iscollected and analyzed following standard protocols [30, 39, 51],following the manufacturers instructions. It is expected that either thedirect conjugation of FITC or detection using a biotin/strepavidin-FITCmay be used to detect the number of OFA/iLRP positive cells inperipheral blood, cell suspension, or other source. The overall numberof OFA/iLRP positive cells can provide insight into aggressivenessand/or progression of the OFA/iLRP positive diseases.

Alternative applications of this method can also be used to determinethe metastatic load of breast cancer through the analysis of sentinelnodes. Briefly, sentinel nodes (or any lymph node close to the initialcancer lesion) are gently disrupted in 5 ml of staining buffer followingstandard protocols. The cell slurry is filtered through a 100 μm mesh,and washed about two times as above. The resultant cell suspension isused to quantify localized metastasis.

The Antibodies can Displace OFA/iLRP or Other Proteins Bound to OFA/iLRP

The location and design of the FREPRLLWTDRADHQPLT antibodies allow forthe possibility that the antibody can displace the interacting OFA/iLRPprotein. Additionally, antibodies designed against other regions may beused to displace interacting proteins or prevent them from binding. Theinitial concentrates on the displacement of or the prevention of thehomodimeric interaction of OFA/iLRP.

To test the possibility that the antibody can displace interactingOFA/iLRP molecules, two separate experiments are performed. First,OFA/iLRP dimer is purified following standard protocols that purify thedimer form of OFA/iLRP. The dimeric form of the protein is incubatedwith increasing concentrations of antibodies against the OFA/iLRP. Afterincubating 1 hour at 37° C. in standard antibody binding buffer (PBScontaining NP-40, Tween-20, BSA, BGG, or other additives), theconversion of dimeric to monomeric forms by antibodies is determined byrunning on a non-denaturing polyacrylamide gel following standardprotocols [37, 38].

A second method that can be used exploits putative changes influorescent polarization depending on molecular size. In the presence ofthe dimer, the primary FP is derived from antibody rotation alone. Ifthe antibody replaces the dimer, there is a shift to monomer and thefluorescent polarization should change.

Fluorophores may additionally be used to quench or modify the emissionspectra due to proximity. Two or more antibodies may be used to modifyemission spectra or quench can be used to determine the state and/orstability of the OFA/iLRP molecule.

The Antibodies are Used as a Cancer Treatment

The antibodies designed to target the OFA/iLRP sequences can be used asa form of treatment or possible prevention for OFA/iLRP positivediseases. For example, testing for the prevention or treatment ofOFA/iLRP positive cancers could occur using similar animal model usingstandard cell lines. To test for the prevention of cancer OFA/iLRP,antibodies can be injected into a rodent (mouse or rat) prior to cancerchallenge. To test for the treatment, OFA/iLRP antibodies can beinjected after the cancer challenge. The cancer challenge is a range ofdifferent known cancerous cell lines, some adherent and somenon-adherent. The cells are grown and introduced into a rodent modelsystem. The amount of OFA/iLRP positive cancer cells can be quantifiedthrough a range of different methods. Additionally, adherent cellsinjected through the tail vein will have increased colonization of thelungs. The treatment and/or preventative abilities of the OFA/iLRPantibody can be calculated by the number of cancer cells present in thelungs of treated versus untreated animals. Additional controls may bethe injection of non-specific isotype control antibodies at the sameschedule as the OFA/iLRP antibodies. Additionally, the OFA/iLRPantibodies augment current cancer therapies. To decrease possibleallergic reactions, the antibodies may be cloned and humanized followingstandard protocol prior to use in humans.

The Antibodies can Alter Biological Function, Leading to Altered EffectsCaused by OFA/iLRP Expressing Related Diseases

The antibodies may alter the overall invasiveness or ability to bind toa standardized substrate. Standard cell lines can be tested followingstandard protocols. One test that is performed to determine the possiblepharmacological affect on mammalian and non-mammalian cells is theeffect on growth rate, for example, the growth of cells with and without the peptides at a range of different concentrations, and to measurethe affect on apoptosis, necrosis, and cell proliferation. OFA/iLRPpositive cancer cells can be grown in vitro on a range of differentbasement membranes with the peptides at a range of doses. The effect ofthe peptides can be measured by a range of different methods including,but not limited to, DNA ladder, cell death detection ELISA, caspasemeasurement, TUNEL assay, Annexin-V membrane alterations, DNA stain,FAS, p53, cytotoxicity assay, cell proliferation, and cell viabilityfollowing standard methods.

The peptides may have the ability to increase or decrease theinvasiveness of a OFA/iLRP positive cancer cell. This can be measured bygrowing OFA/iLRP positive cells with and without a range of differentconcentrations of the peptide using a modified Boyden-chamber similar toseveral studies involving other proteins [52-55]. The peptides may alsoaffect cell adhesion and can be measured using standard methods.Adherent cultured OFA/iLRP positive cancer cells are cultured in thepresence of different extra-cellular matrix proteins (ECM) and with thepeptides. The cells are then assayed, following standard methods todetermine the relative attachment of the cell lines in the presence ofthe peptides [56-59]. Several other commonly used techniques may beapplied to determine the affect of OFA/iLRP on cell viability,proliferation, cell death, and apoptosis [37-39, 42].

The Antibodies are Linked to Colloids to have Anti-Cancer Activity or toProvide an In Vivo Marker for Cancer

The antibodies can be attached or cross-linked to a range of differentcolloids. For example, gold or silver colloid can be used to identifyOFA/iLRP positive cells through resonant light scattering or alternativemethods. Additional uses for the colloid would be one that oxygen-freeradicals or similar can be induced. The colloidal agent linked to anantibody increases the probability and OFA/iLRP positive cancer cell isexposed to free radicals.

The Antibodies are Linked to an X-Ray Opaque Dye, or Similar Type ofConjugate to be Used with an X-Ray

The antibody is conjugated to a radiopaque dye or similar [60]. Theability to detect a cancerous state early and accurately can help withpatient treatment options. It may be possible to conjugate the antibodyto a radiopaque dye or equivalent to increase the efficiency of currentx-ray based techniques. Additionally, the antibody may be linked to amolecule that could be “tuned” to specific resonant frequencies to bedetected by MRI. Possible conjugates include, but are not limited to, achemically reactive gadolinium-based system that can be attached toantibodies similar to used for conjugation to fluorescent dyes. Acommonly used technique is the addition of a tetrafluorophenyl (TFP)ester moiety that reacts with the primary amines found on antibodies.The unreacted TFP gadolinium can be removed using sized exclusionchromatography. In order to be used clinically in humans, the antibodymay need to be humanized. The method described above would followstandard protocols.

As a proof of concept, there is no need for the animal model to have anactive immune system, which allows for the use of standard techniques[61]. The initial work does not need to use x-rays but needs todemonstrate the ability of the OFA/iLRP antibodies to bind in vivo. Theantibody or equivalent is conjugated to a fluorophore, colloid, enzymeconjugate, or other method that can be easily detected. The conjugatedantibody is injected into the animal at a range of different doses, andat defined time-points, the induced tumors can be sampled to look forbinding of the antibody. This binding can show that this can be used totarget tumors for diagnostic imaging. Additional methods include the useof gold nanoparticles that can be coated with epoxy silane derivativesor similar for covalent linkages to the antibodies [62].

The Antibodies are Linked to, and Conjugated with, ChemotherapeuticAgents and Directed to OFA/iLRP-Positive Cancer Cells

In order to decrease the number of side effects associated withclassical chemotherapy, a targeted approach may be taken. The use of theOFA/iLRP antibodies as a targeting agent for anti-cancer drugs mayincrease safety and efficacy of current therapies. A way to test theeffectiveness of this and as a proof of concept, antibodies may beincorporated in a liposomal type system where the liposome contains achemotherapeutic agent. To show targeting ability, OFA/iLRP cells, amixture of OFA/iLRP cancer cells and normal human cells can be mixed ina cell culture plate. The mixture can be added and compared to thetreatment of cells with similar dose of chemotherapeutic agent orliposome with isotype control antibodies incorporated. It is expectedthat the target approach would have a higher mortality of OFA/iLRPpositive cells without as drastic an affect on normal cells. The effectcan be determined through a range of different standard techniques.While standard chemotherapeutic agents can be used, another techniquecould use the attachment of radioactive molecules to the OFA/iLRPantibody. For example, the antibody is iodinated following standardprotocols.

In addition to linking directly to the chemotherapeutic agent, creatinga liposome with the antibodies agent, or a polyacrylamide/agarose beadsoaked in chemotherapeutic agent, the antibody can be attached to analternative delivery method. It may be possible to have carbonnano-tubes or equivalent that can carry chemotherapeutic agents. Wheninteracting with the OFA/iLRP positive cell, a signal could be providedto cause the nano-tubes to resonate and release the containedanti-cancer agent. The trigger can be a range of different methods. Theantibodies can be linked to, and conjugated with, nano-carbon,nano-gold, and other nanotubes and nano-agents, and subjected to amicro-electro/radiowave-sensitive device or similar device in order totarget OFA/iLRP-positive cancer cells. When directly linked between theantibody and the anti-cancer agent, a reactive linker molecule istargeted to release the anti-cancer agent. An example of this could be alinker between the antibody and the anti-cancer agent that is UV orx-ray cleavable. In the presence of x-rays (radiation therapy), thelinker can cleave and release the anticancer drug. Another method uses apeptide linker with a protease cleavage site to be cleaved when in thetumor micro-environment.

The Antibodies can be Linked to, and Conjugated with, Other Proteinsthat Alters the Biological Function of the Target Cells

The antibody can only direct certain types of immune responses. Thefusion of two distinct sequences to create DNA vaccinations have beenused before [63]. This molecule is a hybrid of the OFA/iLRPantibody-binding region that is fused to a biologically active sequencethat can modulate the tumor or surrounding area. A couple ofpossibilities are to clone the humanized OFA/iLRP antibody active regionand fuse it to an immune system regulator, cell cycle regulator, orapoptosis-inducing protein sequence. To prevent non-specific events, theprotein is engineered to remain inactive until in the tumormicro-environment. An OFA/iLRP antibody is fused to a protease cleavagesite linker that is then attached to an apoptosis-inducing peptide or asmall molecule to induce an immune response against the tumor. Thefusion of two different molecules could allow for a multivalent antibodythat can be targeted with increased specificity to cancer cells.

The Use of OFA/iLRP Antibodies with Microelectrofluidic or EquivalentDevices are Used Diagnostically or as an Ongoing Screening Method

The OFA/iLRP antibodies or active regions are coated ontomicroelectrofluidic devices to measure real-time OFA/iLRP expression inthe blood stream. This device is self-contained to act as a monitoringdevice for cancer reassurance. The antibodies are coated on a film orother micro-device to allow for the detection of the cancer. It ispossible to determine the amount bound based on a couple of methods.First, the antibody can be coated on a molecular cantilever that has adefined flow across it. As the real-time interaction between the antigenand antibody occur, there should be a increased torque on the coatedlever. This, in turn, can be measured through changes in resistance orinduction of electrical charge. A similar technique is used with a thinfilm coated with OFA/iLRP antibodies and when bound by the antigen,either the charge or interaction or change of tension on the film can bemeasured and correlated to changes in OFA/iLRP expression. Thesetechniques are not limited to OFA/iLRP, but any cancer molecule can beused as a micro-implanted device that can be read by clinicians asneeded to alert them to reoccurrence or metastatic disease progression.

The Use of OFA/iLRP Antibody Attached to Radioactivity, Fluorescent orEquivalent Molecules to Aid the Medical Doctor in Excision of the Tumor

As an aid to the surgeon during the excision of cancer, the use ofOFA/iLRP antibodies are linked to a fluorophore, radioactive marker, dyeor equivalent, allowing the surgeon to easily identify the cancerousgrowth. Additionally, this technique may be used to quickly identifylymph nodes that may contain metastatic disease, for example, ananti-cancer antibody linked to an appropriate conjugate to allow foridentification, such as current radioactivity is used for sentinel nodebiopsy. After the injection, the patient is brought to surgery where theprocedure occurs as usual. However, if it is difficult to find thecancer or to check for lymph nodes, a Geiger counter, UV, or other lightsource can be used which allows for rapid identification of thecancerous areas. The cancer is excised using the appropriate surgicaltreatment. This method can greatly aid in the identification/excision ofcancer.

The Use of the Antibodies for Immunoprecipitation

The OFA/iLRP antibodies are used for immunoprecipitation to identifynovel therapeutic targets. Cell lysate is immunoprecipitated followingstandard protocols [37, 39]. The resultant proteins can be analyzed onSDS-PAGE, 2d gel electrophoresis, mass spectroscopy, or other methods.The resultant information may provide insight into possibleOFA/iLRP-related mechanisms that can be used for clinical diagnostic ortreatment.

The following examples are intended to illustrate, but not to limit, thescope of the invention. While such examples are typical of those thatmight be used, other procedures known to those skilled in the art mayalternatively be utilized. Indeed, those of ordinary skill in the artcan readily envision and produce further embodiments, based on theteachings herein, without undue experimentation.

Example 1 Method of making OFA/iLRP monoclonal antibodies 2C6 and 3G7

OFA/iLRP monoclonal antibodies 2C6 and 3G7 were made at PrecisionAntibody using standard methods and protocols. The peptide ofFREPRLLVVTDPRC was used to generate monoclonal antibody 3G7. The peptideof YRDPEEIEKEEQC was used to generate monoclonal antibody 2C6. Thesynthesized peptides were conjugated to maleimide-activated KLH usingstandard protocols (Pierce/Thermo, Rockfork, Ill.). Once conjugated, theKLH-OFA peptides were used to immunize two different mice per peptide.After immunization, the serum was obtained via tail bleed and the serumwas used to screen for antibody titer against BSA-conjugated peptides(FIG. 1). If positive against the peptide, an indirect ELISA usingrecombinant human OFA/iLRP was done to verify that that the antibodieswould react with the protein. This was done to ensure that the antibodywould react against small peptides as well as the full-length protein.After the reactivity against the peptide and protein was verified, thespleen cells were fused with a myeloma cell line and selected in 96-wellculture plates. Viable colonies were kept alive and then screenedagainst the peptide and OFA/iLRP (similar to above). The hybridomatissue culture supernatants that had high activity against OFA/iLRP butnot against BSA-coated wells were chosen for immunoglobulinclassification (IgG or IgM). Any clones that were IgM were excluded fromthe screen and the IgG-producing hybridomas were grown followingstandard protocols. To further purify the antibodies from the medium, aprotein G selection was done following standard protocols(Pierce/Thermo, Rockford, Ill.). The eluted antibodies concentration wasdetermined using A260 absorbance and this was used for 2C6 and 3G7antibody production.

Example 2 Analysis of Tail Bleeds for Activity Against rHU OFA

In this study, activity against recombinant full-length human OFA/iLRPwas analyzed. Nunc-Immunostar MaxiSorp plates were coated with OFA/iLRPprotein at a concentration of 10 μg/ml in PBS with sodium azide. Plateswere incubated at 37° C. for 2 hours. Plates were washed with water 3times, then blocked for at least an hour with 5% non-fat dry milk (NFDM)in PBS-t. Plates were washed 3 times with PBS-t tail bleeds and positivecontrols were added in 5% NFDM. Plates were incubated overnight at 4° C.with shaking. Plates were washed as above and incubated for 1 hour atroom temperature in universal secondary antibody diluted 1:100 in 5%NFDM. Plates were washed and incubated for 30 minutes at roomtemperature with Streptavidin-HRP (1:200 R&D systems). Plates werewashed and 200 μl SIGMA FAST™ OPD was added to each well. Plates wereobserved for reaction and the plate was stopped with 50 μl of stop (2nH₂SO₄) while there was still low background. Plates were read andanalyzed on a SpectraMax 384 following the standard protocol on theinstrument for basic endpoint ELISA w/OPD and acid stop.

All of the immunizing peptides designed against OFA/iLRP (Table 2)produced tail bleed serum that can react with the recombinantfull-length OFA protein. When compared to previously developedmonoclonal antibodies provided by Drs. Coggin, Rohrer and Barsoum(Positive control), the tail bleed serum performed similar or slightlybetter (FIG. 2) despite the monoclonal antibody being at a concentrationgreater than 2 mg/ml. This experiment further demonstrates the use ofOFA/iLRP as a capture antigen to determine antibody titre of patient anddiagnostic samples.

The results show that peptide-derived antibodies against OFA/iLRP canrecognize the recombinant full-length protein. Additionally, coating ofthe recombinant full-length OFA onto a plate can be used as a screeningmethod for further antibody development or monitoring of anti-OFA immuneresponses.

Example 3 Indirect ELISA of rOFA/iLRP to Determine the Reactivity ofMonoclonal Antibodies

In this study, the immunoreactivity of monoclonal antibodies designedagainst specific regions of OFA/iLRP to the recombinant OFA/iLRP wasdetermined. Briefly, Immulon 4HBX plates were coated with OFA/iLRPprotein at a concentration of 2 μg/ml in PBS. Plates were incubated at4° C. overnight. Plates were washed with water three times, then blockedfor at least an hour with 1% BSA in PBS-t. Plates were washed threetimes with PBS-t and a 5-fold dilution series of the monoclonalantibodies was run along 8 wells with a high concentration of 1:10 and alow of 1:781,250. Plates were incubated overnight at 4° C. with shaking.Plates were washed as above and incubated for 1 hour with biotinylatedanti-mouse IgG secondary at 1:50,000. Plates were washed and incubatedfor 30 minutes at room temperature with Streptavidin-HRP (1:250,000).Plates were washed and 100 μl TMB Two Component HRP microwell substrate(BioFX Laboratories, Owings Mills, Md.) was added to each well. Plateswere observed for reaction and the plate was stopped with 50 μl of stop(2n H₂SO₄) while there was still low background. Plates were read andanalyzed on a SpectraMax following the standard protocol on theinstrument for basic endpoint ELISA w/HRP and TMB.

The results in FIG. 3 show a dilution curve of the monoclonal antibodies2C6 and 3G7 run on an indirect ELISA against rHu OFA/iLRP (n=4). An8-point dilution series with 5-fold dilutions starting at 1:10 andranging down to 1:781,250 show that there is a strong reaction of themonoclonal 2C6 to the OFA and there is a dynamic range of detectionusing the 2C6 antibody in this assay.

FIG. 3 also shows a dilution curve of the monoclonal antibody 3G7 run onan indirect ELISA against OFA/iLRP. An 8-point dilution series identicalto that above shows that there is strong reaction of the monoclonal 3G7to the OFA and there is a dynamic range of detection using the 3G7antibody in this assay (n=4).

This data indicates that both of these monoclonal antibodies (2C6 and3G7) have the ability to recognize and bind to the full-length OFA/iLRP.This data demonstrates that the engineered antibodies can be used todetect OFA in a specific manner that can be used for a wide range ofdownstream diagnostic tests. They also can be used as standards to lookfor the presence of anti-OFA antibodies in a range of biofluids.

Example 4 Sandwich ELISA of rOFA/iLRP to Determine the Reactivity ofMonoclonal Antibodies

Immulon 4HBX plates were coated with 2C6 monoclonal antibody at aconcentration of 10 μg/ml in PBS. Plates were incubated at roomtemperature overnight. Plates were washed with water three times, thenblocked for an hour with 1% BSA in PBS-t. Plates were washed three timeswith PBS-t and a 2-fold dilution series of OFA/iLRP was run along 8wells with a high concentration of 5000 ng/ml and a low concentration of78.125 ng/ml. OFA/iLRP standards were prepared in 5% BSA in PBS tosimulate serum concentrations. Plates were incubated 2 hours at roomtemperature with shaking. Plates were washed as above and incubated for2 hours with biotinylated 3G7 monoclonal antibody. Plates were washedand incubated for 30 minutes at room temperature with Streptavidin-HRP(1:200) (R&D). Plates were washed and 100 μl of TMB Two Component HRPmicrowell substrate (BioFX Laboratories, Owings Mills, Md.) was added toeach well. Plates were observed for reaction and the plate was stoppedwith 50 μl a of stop (2n H₂SO₄) while there was still low background.Plates were read and analyzed on a SpectraMax at 450 nm following thestandard protocol on the instrument for basic endpoint ELISA w/HRP andTMB.

FIG. 4 shows a standard curve of a wide range of concentration ofOFA/iLRP and the ability of our monoclonal antibodies to detect it usinga sandwich ELISA. This data shows that both of these monoclonalantibodies (2C6 and 3G7) have the ability to recognize and bind to thefull-length OFA/iLRP in a manner that can be detected using sandwichELISAs. When reversed (i.e., 3G7 caputure/2C6 detection), a similarresult was seen but the reaction absorbance was significantly lower thanwhen using the 2C6 as a capture antibody (date not shown). Likewise,this data demonstrates that the engineered antibodies can be used todetect OFA in a specific manner that can be used for a wide range ofdownstream diagnostic tests. This type of ELISA can be used to look forthe presence of OFA/iLRP in a range of biofluids and tissue lysates.Additionally, this technology may be applied for inclusion/exclusioncriteria for OFA/iLRP-based therapies or used as a cancer diagnostic.The antibodies were designed against conserved regions of the proteinand should cross-react with all species that express OFA/iLRP.

Example 5 Fluorescent Polarization of Oncofetal Antigen Immature LamininReceptor Using Peptide Derived Monoclonal Antibodies

This experiment demonstrates the usability of the 2C6 clone of theOFA/iLRP in fluorescent polarization. To simulate a serum sample, allstandards were diluted in 5% Bovine Serum Albumin (BSA) in phosphatebuffered saline. The experiments were based in part on the HSP 90 work,but a fluorsceine-5-maleimide-labeled antibody was used instead ofgeldanamycin-BODIPY, a small fluorescent molecule that can bind HSP 90[49, 45].

OFA/iLRP monoclonal antibodies 2C6 and 3G7 were labeled withFluorescein-5-Maleimide (Pierce/Thermo, Rockford, Ill.) followingstandard protocols. The unbound dye was removed using standard dyeremoval columns following the manufacturer's protocols. After labelingthe antibody, it was used in fluorescent polarization experiments.

OFA/iLRP was diluted to 40000 ng/ml in 5% BSA in PBS and 50 μl was addedto the top well and diluted 6-fold in 5% BSA in PBS in a 96-well blackplate (Cliniplate, Thermo Scientific). A high concentration of serumalbumin (>5% serum albumin) was used to simulate serum concentrations.The labeled antibody was diluted to 20 μg/ml in PBS with 0.1% normalhuman plasma and 0.01% Tween-20. The diluted antibody solution (50 μl)was dispensed into the wells, mixed, sealed, covered from light andincubated overnight at 4° C. The plates were allowed to adjust to roomtemp and the fluorescent polarization was measured on a DTX-880 (BeckmanInstruments, Palo Alto, Calif.). The manufacturer fluorescent protocolwas used except fluorescent integration time of 0.0001 s. The data wasexported to excel and mP was calculated as outlined previously [49]. Theraw data was plotted on a log x-axis (FIG. 5A) using Prism 5.0 (GraphPadSoftware, Inc), analyzed using one-way ANOVA to compare the backgroundto the calculated mP values, and transformed to allow for afour-parameter logistic (FIG. 5B) (r²=0.975) n=2.

The concentrations of OFA in 5% BSA/PBS ranged 40000 to 0.85 ng/ml andshowed a range from less than 1000 ng/ml (due to saturation) to lessthan 30.86 ng/ml (FIG. 5A). At concentrations above 1000 ng/ml, thesignal became saturated, and below 30.6 ng/ml (5.1 ng/ml), the signalwas not significantly different than the background when the data istransformed and a four-parameter logistic is performed and r2 >0.95(FIG. 5B).

The results demonstrate that the peptide-derived OFA/iLRP antibodies canbe used to determine OFA/iLRP concentration using fluorescentpolarization. This data provided by the standard curve had an acceptabler² value that can be used to calculate unknowns from serum, plasma,tissue lysate, or any soluble source contain OFA/iLRP.

Example 6 Immunohistochemical Staining of OFA/iLRP

The purpose of this study was to analyze the ability of our monoclonalantibodies to detect OFA/iLRP in formalin-fixed paraffin-embedded(FFPE), 6 micron thick tumor sections with limited reaction in normaladjacent tissue.

Experiments were performed following the protocol provided with theMaxTag Histo kit for use with mouse Primary Antibody (RocklandImmunochemicals, Gilbertsville, Pa.). Briefly, FFPE 6 micron tumorslides were de-paraffinized with xylenes and then rehydrated withdecreasing concentrations of ethanol (EtOH), then diluted with water,and finally placed in PBS. All incubation steps were carried out in ahumidified chamber. The area on the slide containing the tissue wasmarked with a PAP pen, and then blocked for 5 minutes in 1% hydrogenperoxide and 1% normal goat serum in PBS to remove endogenous peroxidaseactivity. The slides were then washed three times in PBS for 5 minutes.The primary antibody was diluted 1:250 in PBS+1% normal goat serum andincubated on the slide at 4° C. overnight. The previous experiments wererun at primary antibody dilutions of 1:10 and 1:50, and had too high ofa background stain. (Data not shown). The slides were then washed threetimes for five minutes in PBS, then the secondary antibody provided inthe kit was added, and they were incubated for 30 minutes at roomtemperature. The slides were washed again and the diluted streptavidinperoxidase reagent from the kit was added and incubated for 30 minutesat room temperature. The slides were washed again and the supplied DABreagent was added and incubated for 15 minutes while monitoring forcolor development. The DAB reaction was allowed to incubate 10 minuteslonger than the manufacturer's recommendations to determine the truebackground staining of the normal adjacent tissue. The slides were thenwashed three times for two minutes with distilled water. Hematoxylincounterstain was added and the slides were incubated for five minutes.The slides were washed again three times for two minutes with water,dehydrated with 100% EtOH four times for two minutes each, and thencleared with four changes of xylene for two minutes each. The tissueswere observed under a microscope and evaluated with a positive reactionbeing visualized as a brown precipitate and nuclei staining light blue.

The stains were run on multiple tumor types. The slide shown in FIG. 6is a representative section that shows breast invasive ductal carcinoma,moderately differentiated, T2N1M0, that has been stained with the 3G7monoclonal antibody as the primary antibody. Staining of the slideranges from very dark, black precipitate to very light, similar toisotype control run slides (not shown). OFA/iLRP expression has beenseen in all tumor types tested to date.

The results show that the monoclonal antibodies are amenable to IHCstaining protocols and may be used in a range of different experimentaland diagnostic applications. One potential application is forapplicability for OFA/iLRP therapy or as a screening/diagnostic test.Since OFA/iLRP is specifically designed against conserved regions, theseantibodies should cross-react with all species.

Example 7 The Effect of OFA/iLRP Monoclonal Antibodies on Cell Viability

The goal of this experiment is to determine if the monoclonal antibodiesdesigned against the OFA/iLRP have any affect on cell viability. Due tothe nature of OFA/iLRP, it was expected that monoclonal antibodies thatare designed to disrupt the OFA|OFA to LR conversion or that inhibitother protein|protein interactions that will have activity.

All cells were grown in RPMI 1640 with L-glutamine; 100 I.U. Penicillin;100 μg/ml Streptomycin and 10% fetal calf serum at 37° C. in a humidchamber (Mediatech, Inc. Manassas, Va.). DU 145 cells were obtained fromAmerican Type Culture Collection (ATCC, Manassas, Va.) and grown inmedia following standard protocols. DU145 cells were grown to between 75and 85% density and collected following standard protocols and countedusing a modified Neubauer brightline hemacytometer and suspended at400,000 cell/ml.

Monoclonal antibodies (2C6 and 3G7) were dissolved in complete medium.After being diluted to appropriate concentration, 50 μl was dispensedinto a 96-well assay plate, either coated with laminin/entactin complex(50 μg/ml) or untreated (Black with clear bottom) (Corning LifeSciences, Corning, N.Y.), and 50 μl of cells (20,000 cells/well) grownovernight. 20 μl of CellTiter-Blue ((Promega, Madison, Wis.)) was addedto the cells and they were incubated for an additional two hours at 37°C. The cells were read on a DTX-880 (Beckman Inc.) following standardfluorescent protocols with an integration time of 0.001 sec. Todetermine if caspase activity was induced, Caspase 3/7 activity wasdetermined using ApoOne assay (Promega, Madison, Wis.). The data wasexported to Excel and then to Prism 5.0 (GraphPad Software, Inc), whereit was plotted, and analyzed for statistical differences between thebackground controls (diluents used for the peptide) using a one-wayANOVA. To determine the difference between the control group (0) and thetreatments, a Dunnets post test was performed. Any p<0.05 (*) wasconsidered to be significant.

All of the DU145 cells grew on either the uncoated orLaminin/Entactin-coated 96-well plates. In the presence of either 2C6 or3G7, there was an affect on cell viability (FIGS. 7 A and B). Whenstatistically analyzed by a one-way ANOVA, the 2C6 and 3G7 antibodieswhile at higher concentrations, caused a decreased viability whencompared to the control group. However, considering the concentrationdifferences between 3G7 (0.32 mg/ml) and 2C6 (1.29 mg/ml), 3G7 appearsto have greater activity. No significant difference was seen in anygroup in the ApoOne Caspase 3/7 assay.

When DU145 cells were grown in the presence of either antibody, itappears to have had a significant affect on cell viability when comparedto PBS alone. There was no Caspase 3/7 activation and no significantchange in ApoOne assay was seen. This indicates that OFA/iLRP antibodiesmay have the ability to be used as an anticancer therapy.

Many modifications and variations of the invention as herein before setforth can be made without departing from the spirit and scope thereof,and therefore only such limitations should be imposed as are indicatedby the appended claims.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

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TABLE 1 List of different putative epitopes for anti- body generationPossible Antigenic Regions for Development of OFA/iLRP antibodiesInitial Peptides of Interest ALDVLQM DVLKFLAAGT EQYIYKFREPRLLVVTDPRADHQPLT GIYIINL DHQPLTEASYVNLPTIALCNTD KLLLAARAIVAIEGVQVPSVPIQQF PADVSVISS ATPIAGR PLRYVDIAIPC FREPRLLVVTDPR AHSVGLVVTDRADHQPLT MPDLYFYR VVTDPRADHQP APEFTAAQPEVA Actual Used FREPRLLVVTDPRVTDPRADHQPLTE YRDPEEIEKEEQ FPTEDWSAQPATED For Conjugation:FREPRLLVVTDPRC CVTDPRADHQPLTE YRDPEEIEKEEQC CFPTEDWSAQPATED PreviouslyPublished Epitopes TEDWSA TPGTFNQIQAAFREPRLLV SGALDVLQAAGTHLGGTNLDFQMEQYIY DGIYIINLKRTWEKLLLAAR AIVAIENPADVSVISSRNTGQRAVLKFAAATGATPIAGRF TPGTFTNQIQAAFREPRLLV ALCNTDSPLAYVDIAIPCNNIPCNNKGAHSVGLMWWMLAR AAAEKAVTKEEFQGEWTAPA QFPTEDWSAQPATEDWSAAP

TABLE 2 Immunizing peptides designed against OFA/iLRP antibodies Peptide1 FFREPRLLVVTDPRC Peptide 2 CVTDPRADHQPLTE Peptide 3 YRDPEEIEKEEQCPeptide 4 CFPTEDWSAQPATED

1. An isolated antibody that specifically binds to a region of theoncofetal antigen/immature laminin receptor protein (OFA/iLRP), whereinthe region is immunogenic.
 2. The isolated antibody of claim 1, whereinthe immunogenic region is located in the dimerization region ofOFA/iLRP.
 3. The isolated antibody of claim 1, wherein the region ofOFA/iLRP comprises a polypeptide sequence of (a) FFREPRLLVVTDPR, (b)VTDPRADHQPLTE, (c) YRDPEEIEKEEQ, or (d) FPTEDWSAQPATED.
 4. The isolatedantibody of claim 1, wherein the region OFA/iLRP is conjugated to apeptide that increases an immunogenic response.
 5. The isolated antibodyof claim 1, wherein OFA/iLRP is conjugated to Keyhole Limpet hemocyanin,ovalbumin, or serum albumin.
 6. The isolated antibody of claim 1,wherein the antibody is a monoclonal antibody.
 7. The isolated antibodyof claim 6, wherein the monoclonal antibody is 2C6 or 3G7.
 8. Theisolated antibody of claim 6, wherein the region comprises thepolypeptide sequence of FREPRLLVVTDPRC or YRDPEEIEKEEQC.
 9. Apharmaceutical composition comprising the antibody of claim
 1. 10. Thecomposition of claim 9, wherein the antibody is a monoclonal antibody.11. The composition of claim 10, wherein the monoclonal antibody is 3G7or 2C6.
 12. The composition of claim 9, further comprising apharmaceutically acceptable carrier.
 13. A method of detecting OFA/iLRPin a sample comprising: (a) contacting the sample with a first and asecond antibody that specifically bind to a region of OFA/iLRP, whereinat least one of the antibodies is specific for OFA/iLRP; (b) allowingthe antibodies to bind to OFA/iLRP and form a sandwich with OFA/iLRP;and (c) detecting the expression of OFA/iLR in the sample using theantibody specific for OFA/iLRP.
 14. A method in accordance with claim13, wherein one of the antibodies binds to both OFA/iLRP and mature LRP.15. A method of claim 14, wherein the antibody recognize both OFA/iLRPand mature LRP acts as a capture antibody, and the antibody specific forOFA/iLRP acts as a detection antibody.
 16. A method of claim 13, whereinthe first antibody is 3G7, and the second antibody is 2C6.
 17. A methodof detecting cancer in a sample comprising: (a) contacting the samplewith antibodies specific for OFA/iLRP; (b) contacting the sample with abiotinylated secondary antibody; and (c) detecting OFA/iLRP in thesample using streptavidin, wherein the detection of OFA/iLRP in thesample is indicative of cancer.
 18. A method of determining the amountof OFA/iLRP in a sample comprising: (a) conjugating an antibody specificfor OFA/iLRP to a fluorophore; (b) contacting the conjugated antibodywith sample; and (c) determining the amount of OFA/iLRP in the sampleusing fluorescent polarization.
 19. A method of determining the amountof OFA/iLRP positive cancer cells in a blood sample comprising: (a)contacting a blood sample with an antibody specific for OFA/iLRP and (b)determining the amount of OFA/iLRP in the sample using flow cytometry.20. A method of treating a OFA/iLRP positive cancer comprisingadministering an amount of antibodies specific for OFA/iLRP to a subjectwith the OFA/iLRP positive cancer that are in an amount sufficient totreat the cancer.
 21. The method according to claim 20, wherein theantibodies are linked to colloids having anti-cancer properties.
 22. Themethod according to claim 19, wherein the antibodies are conjugated withchemotherapeutic agents.
 23. The method according to claim 19, whereinthe antibodies are conjugated with proteins.
 24. A method of detecting aOFA/iLRP positive cancer in a subject comprising: (a) conjugatingantibodies specific for OFA/iLRP to a radio opaque dye; (b)administering the conjugated antibody to a subject; and (c) detectingthe conjugated antibody using an x-ray, wherein the detection ofOFA/iLRP on the x-ray is indicative of cancer.